The objective of this research is to discover why cells require insulin for growth. I intend to examine a line of cells in culture unique in that they grow independently of insulin and related growth factors. I hope to define the metabolic mechanism by which these cells overcome the nearly universal dependence of other cells for insulin or "insulin-like" hormones in tissue culture. A subculture of human cells is propagated in tissue culture in defined medium (NCI-H69 small cell lung carcinoma cells). The subculture grows in the absence of insulin and related growth factors, while the parent line also cultured and serving as a control, requires high levels of insulin in its medium to grow. Experiments will focus on: a) Insulin and insulin-like growth factor (IGF) receptors. The binding and competition curves of labeled insulin and IGF's to the subculture and parent lines of the cells will be investigated to determine what are the characteristics of the receptors associated with growth in these cells. b) "Post receptor" metabolic events. Measurement of the stimulation of glucose oxidation, DNA synthesis and protein synthesis in the cells by insulin and the IGF's. The relationship between these biological functions and hormone binding will be determined. c) Autocrine and paracrine secretion: Possible production of substances into the media by the cells themselves that promote their own growth and eliminate the need for added insulin. Conditioned media will be assayed for IGF activity by radioimmunoassay, radioreceptor assay and bioassay. The activities will be purified in gel filtration, ion exchange chromatography and HPLC.